Project Summary Breast cancer remains the most commonly diagnosed cancer for women, and 30% of mortality is attributable to the spread of cancer to the brain in a process called metastasis. When brain metastases are present, women have a five-year survival of only 25%, with a median survival of less than one year. Unfortunately, as new treatments provide better control of primary breast cancer, the incidence of brain metastases is increasing. The greatest challenge in the clinical management of patients who relapse with brain metastasis is the extremely limited treatment options. Systemic treatments, such as chemotherapies or targeted therapies, cannot effectively treat micrometastatic brain lesions or prevent brain relapse, largely due to their inability to penetrate the blood-brain barrier. Currently, no clinically approved drug shows promising efficacy for brain metastases. Therefore, it is urgent that we expand our mechanistic understanding of breast cancer brain metastases so that we can develop new and effective systemic targeted therapeutics for this rapidly expanding patient population. Although thousands of cancer cells can be found in the circulation of patients, the formation of brain metastases is an inefficient and highly selective process. This is due in part to the unique nature of the brain microenvironment, which includes expression of neurotransmitters not found elsewhere in the body. One neurotransmitter pathway, the GABA signaling pathway, is increased in clinical breast cancer brain metastases. Mutual communication between cancer cells and the metastatic environment is essential for the successful formation of metastases. The cellular characteristics of the tumor cell ? both its inherent genetic traits as well as yet to be defined secondary, non-genetic changes influenced by the microenvironment ? dictate its ability to successfully complete the metastatic process. This proposal investigates a novel mechanism of breast cancer adaptation to the brain microenvironment, and seeks to identify how this adaptation subsequently propels metastatic outgrowth. A high throughput experimental methodology employing RNA-seq and RNAi functional screening identified putative mediators of brain metastasis. Rab11, a component of the endosomal recycling pathway, is up-regulated during adaptation to the brain microenvironment, suggesting involvement of recycling in brain metastasis. As Rab11 regulates the specificity of vesicular cargo during endosomal recycling, this proposal hypothesizes that Rab11 mediates breast cancer brain metastatic outgrowth through recycling mediated up-regulation of the GABA receptor. The proposed research will 1) determine the mechanism and consequence of Rab11 up-regulation in brain metastasis; 2) explore the mechanism of Rab11-mediated GABA receptor recycling; 3) investigate the efficacy of targeting the recycling pathway in the treatment of brain metastases.